Immunohistochemical markers for equine granulosa cell tumors: a pilot study.

Sex cord-stromal tumors (SCSTs), generally referred to as granulosa cell tumors (GCTs) or granulosa-theca cell tumors (GTCTs) in equids, show complex compositions and variable numbers of hormone-producing cells. These tumors can be difficult to diagnose, especially in early stages. Therefore, we tested a panel of antibodies for vimentin, smooth muscle actin, laminin, Ki-67, E-cadherin, calretinin, moesin, p-ezrin, AMH, and aromatase, markers used for tumor composition and classification, progression, and prognosis in human SCSTs, on an exemplary grapefruit-size equine GCT within the left ovary of a 13-year-old mare with stallion-like behavior and elevated testosterone levels in comparison with normal ovarian tissue. The tumor showed a low proliferation rate and prominent moesin and p-ezrin staining in granulosa cells. E-cadherin, calretinin, aromatase, and AMH are suggested to be potential markers for different cell components of equine SCSTs that can support tumor diagnosis and classification.

Lipid droplet dynamics in healthy and pyometra-affected canine endometrium.

BACKGROUND: Accumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model. METHODS AND RESULTS: We characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified. CONCLUSIONS: We found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.

Expression of COX-2, p16, and Ki67 in the range from normal breast tissue to breast cancer.

The increasing number of diagnosed breast lesions lead to the critical need for new markers that would elucidate the process of tumorigenesis. The objective of the study was to examine COX-2, p16, and Ki67 expression in a broad spectrum of breast lesions in order to define the proteins' phenotype throughout the tumorigenesis. Expression was studied by immunohistochemistry in 308 human breast samples divided into 7 subgroups - flat epithelial atypia (FEA), atypical hyperplasia (ADH), intraductal carcinoma (DCIS), invasive cancer (IC), benign lesions (BLs), normal tissue adjacent to breast cancer (CANT), and fatty tissue (FT). Analysis among 4 subgroups - premalignant lesions (DIN), IC, BLs, and normal tissue was also performed. High prevalence of COX-2 overexpression was found in all breast lesions including BLs (70% FEA, 89% ADH, 86% DCIS, 81% IC, 44% CANT, 92% BLs, 29% FT). Significant dominance of p16 overexpression was found in premalignant lesions and BLs (50% FEA, 67% ADH, 50% DCIS, 37% IC, 8% CANT, 58% BLs, 21% FT). The location of staining within p16+ cells differed - BLs showed nuclear positivity, whereas in IC it was exclusively cytoplasmic. Premalignant lesions showed all types of p16 positivity. Significantly higher prevalence of COX-2+p16+Ki67+ phenotype was in premalignant tumors with the highest prevalence in ADH (40% of FEA, 67% ADH, 35% DCIS, 20% IC, 3% CANT, 20% BLs, 14% FT). Our observations showed a high prevalence of COX-2+p16+Ki67+ phenotype in premalignant lesions. Further studies are needed in order to elucidate if this phenotype reflects any specific pathway of future progression of premalignant breast lesions.

Long Term Evaluation of Nanofibrous, Bioabsorbable Polycarbonate Urethane Grafts for Small Diameter Vessel Replacement in Rodents.

OBJECTIVE: Biodegradable materials for in situ vascular tissue engineering could meet the increasing clinical demand for sufficient synthetic small diameter vascular substitutes in aortocoronary bypass and peripheral vascular surgery. The aim of this study was to design a new degradable thermoplastic polycarbonate urethane (dPCU) with improved biocompatibility and optimal biomechanical properties. Electrospun conduits made from dPCU were evaluated in short and long term follow up and compared with expanded polytetrafluoroethylene (ePTFE) controls. METHODS: Both conduits were investigated prior to implantation to assess their biocompatibility and inflammatory potential via real time polymerase chain reaction using a macrophage culture. dPCU grafts (n = 28) and ePTFE controls (n = 28) were then implanted into the infrarenal abdominal aorta of Sprague-Dawley rats. After seven days, one, six, and 12 months, grafts were analysed by histology and immunohistochemistry (IHC) and assessed biomechanically. RESULTS: Anti-inflammatory signalling was upregulated in dPCU conduits and increased significantly over time in vitro. dPCU and ePTFE grafts offered excellent long and short term patency rates (92.9% in both groups at 12 months) in the rat model without dilatation or aneurysm formation. In comparison to ePTFE, dPCU grafts showed transmural ingrowth of vascular specific cells resulting in a structured neovessel formation around the graft. The graft material was slowly reduced, while the compliance of the neovessel increased over time. CONCLUSION: The newly designed dPCU grafts have the potential to be safely applied for in situ vascular tissue engineering applications. The degradable substitutes showed good in vivo performance and revealed desirable characteristics such as biomechanical stability, non-thrombogenicity, and minimal inflammatory response after long term implantation.

Expression of enzymes involved in polyunsaturated fatty acid synthesis in the stallion testis and epididymis.

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.

Intussusceptive Pillar Formation in Developing Porcine Glomeruli.

BACKGROUND/AIMS: Intussusceptive angiogenesis (IA) is a dynamic process which contributes to vascular expansion and remodeling. Intraluminal pillars have long been the distinctive structural indicator of IA. However, the mechanism of their formation has not been fully elucidated. METHODS: Using light and electron microscopy, we studied intussusceptive vascular growth in the developing porcine metanephric kidney. RESULTS: We observed intraluminal pillars formed by endothelial cells in the vasculature of developing glomeruli. Their diameter was < 2.5 µm, consistent with the diameter of nascent pillars. TEM revealed that the majority of these pillars consisted only of endothelium. However, a central core of extracellular matrix (ECM) covered by endothelium, reminiscent of a more mature intussusceptive pillar, was also found in the lumen of a glomerular capillary. Perivascular cells or pericytes were not involved in the pillar structure during these stages of formation. CONCLUSION: This study shows ECM presence in a mature intussusceptive pillar without any perivascular cell involvement in the structure. This leads to the hypothesis that ECM deposition precedes the participation of these cells in the formation of intraluminal pillars during IA in porcine metanephric glomerular capillaries.

Alternative lengthening of telomeres does exist in various canine sarcomas.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) found in some human tumors such as sarcomas. Canine tumors are not characterized for ALT and the study aim was to identify if the ALT phenotype exists in canine sarcomas. Sixty-four canine sarcoma samples (20 snap-frozen, 44 FFPE) as well as six canine sarcoma cell lines were screened for ALT by C-circle assay. ALT was further evaluated by measuring telomere length via qPCR and telomere restriction-fragments including pulsed-field electrophoresis. ALT-associated proteins were validated by immunohistochemistry. Further, telomerase activity (TA) and gene expression were analyzed by TRAP and qPCR. DNA from 20 human neuroblastomas and 8 sarcoma cell lines served as comparative controls. ALT was detected in 9.4% (6/64) canine sarcomas including aggressive subtypes as hemangiosarcoma, osteosarcoma, and histiocytic sarcoma. C-circle levels were comparable with human ALT-positive controls. All ALT tumors demonstrated loss of ATRX expression and 5/6 showed strong p53 expression. TA was detected in 93% (14/15) snap-frozen samples including a sarcoma with ALT activity. This tumor showed long heterogeneous telomeres, and a high level of colocalization of DAXX with telomeres. One sarcoma was ALT and TA negative. All canine and human sarcoma cell lines were ALT negative. In this study, we demonstrated that canine sarcomas use ALT. As in humans, ALT was identified in aggressive sarcomas subtypes and coexisted with TA in one tumor. Overall, canine sarcomas seem to share many similarities with their human counterparts and appear an attractive model for comparative telomere research. © 2016 Wiley Periodicals, Inc.

Impact of superovulation and mating on the wellbeing of juvenile and adult C57BL/6N mice.

Superovulation of mice is routinely used to increase the number of obtainable ova per female. Because of the better outcome, prepubescent females are preferentially used. Here, we provide results of the impact of superovulation and mating on the wellbeing of juvenile compared with adult C57BL/6N mice. Two groups of mice (3-4 weeks vs 7-8 weeks old) were superovulated and mated. Observation of mating behaviour showed that reluctant adult females tended to fight the male's approach, whereas juveniles preferred to take flight. Faeces were collected daily for the analysis of stress hormones. There was no difference in the levels of glucocorticoid metabolites either between age groups or between treated animals and their controls. Histology after mating revealed intact vaginal mucosa without any detectable lesions in all animals regardless of age. In contrast to adults, almost all juveniles were synchronised in oestrus and produced significantly more ova. Taken together, our results reveal no increased welfare problem from using juvenile mice for superovulation and mating. Considering the higher yield of fertilisable oocytes and zygotes, it is advisable to use C57BL/6N prepubescent mice in order to reduce the number of donor females required.

Effect of stallion age on the expression of LH and FSH receptors and aromatase P450 in equine male reproductive tissues.

The aim of the present study was to characterise receptors for LH and FSH (LHR and FSHR, respectively) and aromatase in epididymal and testicular tissue from stallions of different ages (prepubertal, young, mature and old). Gene and protein expression were assessed by real-time quantitative polymerase chain reaction (real-time qPCR), immunohistochemistry and multiple immunofluorescence labelling. There were no differences in LHR mRNA expression in epididymal and testicular parenchyma in stallions of different age. In contrast, expression of FSHR and CYP19A1 in caput, corpus and cauda epididymis and in testicular parenchyma increased with age (P<0.001). Immunolabelling for LHR, FSHR and aromatase was influenced by puberty. In postpubertal stallions, positive staining for LHR and aromatase was detected in Leydig cells, whereas protein expression of FSHR was present in Sertoli cells and primary spermatocytes. In prepubertal colts, staining for LHR, FSHR and aromatase was detected in seminiferous tubules. In epididymal tissue, aromatase was present in the cauda epididymis only, regardless of age. In conclusion, the results highlight the significance of gonadotropin action and oestrogen production for the maturation of male reproductive tissue in the horse. The presence of FSHR in the seminiferous tubules suggests effects of FSH on spermatogenesis in this species. The importance of oestrogen production for maintenance of testicular function in stallions was confirmed.

Canine and human sarcomas exhibit predominant FGFR1 expression and impaired viability after inhibition of signaling.

Fibroblast growth factor receptors (FGFRs) are important in malignant progression of several human epithelial tumors. However, little is known about FGFRs in canine or human soft tissue sarcomas. Thus, our aim was to investigate expression of FGFRs and their involvement in cell survival in sarcomas of both species. FGFR1-4 and FGFRL1 transcripts as well as IIIb/IIIc splice variants of FGFR1-3 were evaluated in 3 canine- and 6 human sarcoma cell lines and 19 spontaneous canine sarcomas by SYBRqPCR. FGFR1 protein expression was assessed by immunohistochemistry. Growth inhibitory effects of FGFR1 inhibitor PD166866 and dominant negative recombinant FGFR adenoviral expression constructs (dnFGFR) on tumor cell lines were analyzed. Profiling of multiple FGFR transcripts detected comparable co-expression in most of human and canine sarcoma cell lines and canine tumor specimens. This indicates existence of closely related regulation mechanisms for FGFR expression in sarcomas of both species. FGFR1 with splice variant IIIc was consistently expressed with highest transcript levels. In 88% of the spontaneous tumor samples a heterogeneous FGFR1 protein expression was observed. Significant growth inhibition and cell death was seen after infection with dnFGFR1 in canine and human sarcoma cells, but not with dnFGFR3 and 4. PD166866 showed selective cytotoxicity with IC50 values between 12.1 and 26.4 µM. FGFR1 inhibition blocked ligand-induced tyrosine phosphorylation of ERK1/2 mitogen-activated protein kinase isoforms. This study emphasizes the important role FGFR1, especially splice variant IIIc, likely plays in sarcomas. Inhibitory small molecules could be of potential use for targeted therapy in aggressive sarcomas of both species.

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses.

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal's age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death.

Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time.

Involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares.

Mares resume ovarian activity rapidly after foaling. Besides follicle-stimulating hormone (FSH) and luteinizing hormone (LH), the pituitary synthesizes prolactin and growth hormone which stimulate insulin-like growth factor (IGF) synthesis in the liver. We tested the hypothesis that follicular growth is initiated already antepartum, mares with early and delayed ovulation differ in IGF-1 release and that there is an additional IGF-1 synthesis in the placenta. Plasma concentrations of LH, FSH, IGF-1, IGF-2, activin and prolactin. IGF-1, IGF-2, prolactin and their receptors in placental tissues were analyzed at the mRNA and protein level. Follicular growth was determined from 15 days before to 15 days after foaling in 14 pregnancies. Mares ovulating within 15 days postpartum formed group OV (n=5) and mares not ovulating within 15 days group NOV (n=9). Before foaling, follicles with a diameter >1 cm were present in all mares and their number increased over time (p<0.05). Follicle growth after foaling was more pronounced in OV mares (day p<0.001, group p<0.05, day x group p<0.05) in parallel to an increase in LH concentration (p<0.001, day x group p<0.001) while FSH increased (p<0.001) similarly in both groups. Plasma concentrations of IGF-1 and prolactin peaked one day after foaling (p<0.001). The IGF-1 mRNA abundance was higher in the allantochorion but lower in the amnion of OV versus NOV mares (group p=0.01, localization x group p<0.01). The IGF-1 receptor mRNA was most abundant in the allantochorion (p<0.001) and IGF-1 protein was expressed in placental tissue without differences between groups. In conclusion, follicular growth in mares is initiated before foaling and placental IGF-1 may enhance resumption of ovulatory cycles.

Immunophenotype investigation in feline intestinal non-B-cell lymphoma.

Lymphoma is the most common tumour of domestic cats, developing most frequently in the small intestine. Feline small intestinal lymphoma predominantly demonstrates a T-cell immunophenotype identified by standard immunopositivity for T cells with CD3 or immunopositivity for B cells with CD20. In contrast, a wide spectrum of immunohistochemical antibodies are applied in humans to diagnose the various specific lymphoma subtypes according to the WHO classification. Our aim was to augment our knowledge of immunophenotypes in feline non-B-cell lymphomas forming macroscopic masses in the intestinal tract. We evaluated the combined immunohistochemistry and flow cytometry findings from 15 cases. Neoplastic lymphoid cells were immunopositive for CD3 in 93% (14/15), granzyme B in 87% (13/15), CD5 in 20% (3/15), CD8 in 13% (2/15), CD4 in 7% (1/15) and CD56 in 7% (1/15) of cases. Cytotoxic granules indicating a cytotoxic origin of the neoplastic cells were identified by histopathology only in 13% (2/15) and by cytology in 47% (7/15) of the cases. Without immunohistochemical labelling of the cytotoxic protein granzyme B, the cytotoxic status would have been missed in 46% (6/13) of the cytological and in 85% (11/13) of the histopathological slides. These findings suggest that more complex immunophenotyping may advance our understanding and help prognosticate small intestinal T-cell lymphoma in cats.

Endocrine changes induced by GnRH immunisation and subsequent early re-stimulation of testicular function with a GnRH agonist in stallions.

CONTEXT: Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. AIMS: This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. METHODS: Shetland stallions were assigned to an experimental and a control group (n =6 each). Experimental stallions were GnRH-immunised twice, 4weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4µg/day for 4weeks followed by 8µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. KEY RESULTS: GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. CONCLUSIONS: Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. IMPLICATIONS: Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function.

Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium.

BACKGROUND: Oestrogens and progesterone have a significant impact on the endometrium during the canine oestrous cycle. Their receptors mediate plasma steroid hormone levels and are expressed in several endometrial cell types. Altered steroid receptor expression patterns are involved in serious uterine diseases; however the mechanisms of hormone action during pathogenesis in these tissues remain unclear. The development of 3D culture systems of canine endometrial cells provides an opportunity for the effects of steroid hormones to be quantitatively assessed in a more in vivo-like setting. The present study aimed to determine the effects of the steroid hormones 17ß-estradiol (E) and progesterone (P) on the expression of the oestrogen and progesterone receptors (ER and PR), and on proliferative activity, in a 3D co-culture system of canine uterine origin, comprising differentiated endometrial glands, and stromal cells (SCs). RESULTS: Morphology, differentiation, and apical-basolateral polarity of cultured glandular epithelial cells (GECs) were comparable to those in native uterine tissue as assessed by immunohistochemistry using differentiation markers (ß-catenin, laminin), lectin histochemistry, and transmission electron microscopy. Supplementation of our 3D-culture system with E (at 15, 30 and 100 pg/mL) resulted in constant levels of ER expression in GECs, but reduced expression levels in SCs. PR expression was reduced in both GECs and SCs following treatment with E. 3 ng/mL P resulted in increased ER expression in GECs, but a decrease in SCs. PR expression in GECs increased in all P-treated groups, whereas PRs in SCs decreased with the lowest and highest doses, but increased with the middle dose of treatment. Proliferative activity, assessed by Ki67 staining, remained below 1% in all assays and cell types. CONCLUSIONS: The present study demonstrates the applicability of our 3D organotypic canine endometrium-derived culture system for cellular-level studies. 3D cultures represent near-physiological systems allowing reproducible quantitative experimentation, thus reducing the need to experiment on living animals. The results of the present investigation emphasize the importance of co-culture of the uterine glands with SCs, as it was shown that the responsiveness of the different cell types to steroid hormones were divergent in the 3D cell culture model.

Abundance of Anti-Muellerian hormone in cat ovaries and correlation of its plasma concentration with animal age, weight and stage of the estrous cycle.

In female animals of different species, Anti-Müllerian hormone (AMH) is produced by follicular granulosa cells and has been associated with the ovarian follicle pool. Because concentration of AMH in plasma of ovary-intact female cats is apparently more variable than previously assumed, we have analysed AMH concentration in blood of cats (n = 93) presented for routine ovariectomy and assessed ovarian histology and AMH protein expression in the surgically removed ovaries. We hypothesised that AMH is synthesized only in preantral and small antral follicles and that plasma AMH concentration reflects the antral follicle count (AFC). Corpora lutea were detected in 35% of the female cats, whereas plasma progesterone concentration was ≥1 ng/mL in 57% of the cats. Follicular cysts were present in 15 cats (16%). Positive immunostaining for AMH protein was detected in close to all primordial and antral follicles, ovarian cysts, 70% of corpora lutea and 28% of atretic follicles. Concentration of AMH in plasma averaged 6.8 ± 0.5 ng/mL (range 1.3-21.7 ng/mL). The AFC increased with increasing AMH concentration with a moderate positive correlation between AFC and AMH (r = 0.286, p < 0.01). Plasma AMH concentration was not affected by season or cats' age, weight, stage of the estrous cycle and presence of follicular cysts. In conclusion, AMH protein is expressed in all endocrine structures of the cat ovary. While AMH is a marker for the presence of ovarian tissue, its usefulness to assess ovarian function in individual female cats is of limited value.

Osteopontin and Ki-67 expression in World Health Organization graded canine meningioma.

Osteopontin (OPN) is a matrix protein involved in tumour initiation and progression. In human meningioma, OPN has been correlated with World Health Organization (WHO) grade, brain invasion and recurrence. The aim of this study was to investigate OPN as a possible malignancy marker in canine meningioma by correlating its expression to WHO grade and proliferative activity as measured by the Ki-67 labelling index (LI). Thirty-five formalin-fixed, paraffin-embedded canine meningioma samples were classified according to the current human WHO classification. Evaluation of OPN expression was performed by immunohistochemical (IHC) labelling and calculation of the OPN intensity score (IS), OPN IHC score and Allred score. The scores were compared with WHO grades, Ki-67 LI, location and invasiveness. Nineteen meningiomas were graded as WHO grade I (54.3%), nine as grade II (25.7%) and seven as grade III (20.0%). Twenty-six tumours were located intracranially, four were retrobulbar and five were spinal meningiomas. In all specimens OPN expression was detected in moderate to high degrees. Neither the OPN scores nor the Ki-67 LIs were correlated with WHO grades. However, the OPN IS and OPN IHC score were significantly higher in WHO grade I samples compared with grade II samples (P <0.05). The OPN IS and OPN IHC score were significantly lower in meningioma samples that invaded surrounding tissues (P = 0.01 and 0.019, respectively). The results indicate a generally high expression of OPN in canine meningioma independent of WHO grade. Further research into the role of OPN as a possible therapeutic target or predictor of recurrence is warranted.